An immunoglobulin contains in general two light polypeptide chains and two heavy polypeptide chains. Each of the heavy and light polypeptide chains comprises a variable region (generally the amino terminal portion of the polypeptide chain) which contains a binding domain that is able to interact with an antigen. Each of the heavy and light polypeptide chains also comprises a constant region (generally the carboxyl terminal portion). The constant region of the heavy chain mediates the binding of the immunoglobulin e.g. to cells bearing an Fc gamma receptor (FcγR), such as phagocytic cells, or to cells bearing the neonatal Fc-receptor (FcRn) also known as Brambell receptor, and also mediates the binding to some factors including factors of the classical complement system such as component (C1q).
Hulett and Hogarth (Hulett, M. D. and Hogarth, P. M., Adv. Immunol. 57 (1994) 1-127) reported that the extracellular receptors for the Fc part of immunoglobulins of class G are a family of transmembrane glycoproteins comprising three different receptor types having different binding specificity: FcγRI, FcγRII, and FcγRIII. Receptors of type I interact with non-complexed IgG, whereas receptors of type II and III interact preferably with complexed IgG.
Human FcγRIII (CD 16) exists in two isoforms and two polymorphic forms. The first isoform FcγRIIIa is a transmembrane molecule encoded by a different gene than the second isoform FcγRIIIb, which is a GPI-anchored membrane protein. Polymorphic form V159 has a valine residue at position 159 of the amino acid sequences whereas the polymorphic form F159 has a phenylalanine residue at position 159.
For the IgG class of immunoglobulins, ADCC and ADCP are governed by engagement of the Fc-region with a family of receptors referred to as Fc-gamma (Fcγ) receptors (FcγRs). In humans, this protein family comprises FcγRI (CD64), FcγRII (CD32), including isoforms FcγRIIA, FcγRIIB, and FcγRIIC, and FcγRIII (CD16), including isoforms FcγRIIIA and FcγRIIIB (Raghavan and Bjorkman, Annu Rev. Cell Dev. Biol. 12 (1996) 181-220; Abes, et al., Expert Reviews (2009) 735-747). FcγRs are expressed on a variety of immune cells, and formation of the Fc/FcγR complex recruits these cells to sites of bound antigen, typically resulting in signaling and subsequent immune responses such as release of inflammation mediators, B-cell activation, endocytosis, phagocytosis, and cytotoxic attack. Furthermore, whereas FcγRI, FcγRIIA/C, and FcγRIIIA are activating receptors characterized by an intracellular immunoreceptor tyrosine-based activation motif (ITAM), FcγRIIB has an inhibitory motif (ITIM) and is therefore inhibitory. While FcγRI binds monomeric IgG with high affinity, FcγRIII and FcγRII are low-affinity receptors, interacting with complexed or aggregated IgG.
The binding of IgG to activating and inhibitory Fcγ receptors or the first component of complement (C1q) depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for FcγRs and complement C1q binding, and have unique sequences. Substitution of human IgG1 and IgG2 residues at positions 233-236 and IgG4 residues at positions 327,330 and 331 greatly reduced ADCC and CDC (Armour, et al., Eur. J. Immunol. 29 (1999) 2613-2624; Shields, et al., J. Biol. Chem. 276 (2001) 6591-6604). Idusogie, et al. (J. Immunol 166 (2000) 2571-2575) mapped the C1q binding site for the therapeutic antibody Rituxan® and showed that the Pro329Ala substitution reduced the ability of Rituximab to bind C1q and activate complement. Substitution of Pro329 with Ala has been reported to lead to a reduced binding to the FcγRI, FcγRII and FcγRIIIA receptors (Shields, et al., J. Biol. Chem. 276 (2001) 6591-6604) but this mutation has also been described as exhibiting a wild-type-like binding to the FcγRI and FcγRII and only a very small decrease in binding to the FcγRIIIA receptor (Table 1 and Table 2 in EP 1 068 241, Genentech).
In WO 2010/048313 recombinant FcRn and variants thereof for purification of Fc-containing fusion proteins are reported. The high level expression and secretion of Fc-X fusion proteins in mammalian cells is reported by Lo et al. (Lo, K-M., et al., Prot. Eng. 11 (1998) 495-500). Dumont, F. A., et al. (Biodrugs 20 (2006) 151-160) report monomeric Fc-fusions. Receptor-Fc fusion therapeutics, traps, and MIMETIBODY™ technology is reported by Huang, C. (Curr. Opin. Biotechnol. 20 (2009) 592-599). In WO 01/03737 immunoglobulin fusion proteins are reported. The expression and export of anti-obesity proteins as Fc fusion proteins is reported in WO 00/40615.